Fungal small RNAs ride in extracellular vesicles to enter plant cells through clathrin-mediated endocytosis

Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea-secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.

important for RNA trafficking. Indeed, EVs have been utilized by plants to deliver plant small RNAs to fungal pathogens during cross-kingdom RNAi 2 . Furthermore, many fungal species have been shown to secrete EVs, including yeast and filamentous fungi [27][28][29][30] . Although, only recently have EVs from plant pathogenic fungi such as the maize smut pathogen Ustilago maydis 31 , the wheat pathogen Zymoseptoria tritici 32 , and the cotton pathogen Fusarium oxysporum f. sp. vasinfectum 33 been characterized. Still, it remains unclear whether plant fungal pathogens secrete EVs to deliver sRNAs into interacting plant cells.
Endocytosis is the primary route for eukaryotic cells to internalize plasma membrane proteins, lipids, and extracellular materials into the cytoplasm 34,35 . In plants, endocytosis is involved in many critical biological processes, including growth, development, signal transduction, nutrient uptake, and plant-microbe interactions [36][37][38][39] . In the past decades, substantial progress has been made in characterizing the function of endocytosis in plant immune responses. For instance, several plasma-membrane localized receptor proteins involved in the plant defense response are internalized through the endocytosis pathway 36,[39][40][41] . Analogous to animal and yeast cells, clathrin-mediated endocytosis (CME) is the main route to internalize extracellular and plasma membrane materials in plants 42 . Clathrin is a triskelion-shaped complex composed of three clathrin heavy chains (CHCs) and three clathrin light chains (CLCs) 34 . By interacting with the plasma membrane, cargo proteins, adaptor protein 2 complex (composed of α, β2, μ2, and σ2 subunits) and other accessory protein factors, clathrincoated pits (CCPs) can be formed 34,43 . The CCPs will then bud off from the plasma membrane to form clathrin-coated vesicles (CCVs) to deliver cargos into cells 34 . CME also contributes to the internalization of protein effectors from the fungal pathogen Magnaporthe oryzae and the oomycete pathogen Phytophthora infestans into plant cells 44,45 . In addition to CME, other sterol-sensitive clathrin-independent endocytosis (CIE) pathways exist in plants. The most well-studied proteins in the CIE pathway are membrane microdomain-associated flotillin1 and remorins 46,47 . Whether CME and/or CIE pathways play a role in the internalization of fungal sRNA effectors and EVs is unknown.
Here, we find that B. cinerea secretes EVs to deliver fungal sRNAs, and the B. cinerea tetraspanin protein BcPLS1 is essential for B. cinerea pathogenicity and EV-mediated Bc-sRNA secretion. Furthermore, utilizing genetics, biochemistry, and cell biology approaches, we demonstrate that CME is the major pathway for the uptake of fungal EVs and their sRNA cargo.

Results
B. cinerea can secrete BcPLS1-positive EVs during infection B. cinerea EVs were first isolated from the supernatants of wild-type strain B05.10 liquid culture using the traditional differential ultracentrifugation (DUC) method 27 . Nanoparticle tracking analysis (NTA) was used to determine the size distribution of isolated EVs, and a peak at 113 nm was observed for B. cinerea EVs (Supplementary Fig. 1a). The isolated EVs were further examined by transmission electron microscopy (TEM), which confirmed the typical cup-shaped form of EVs ( Supplementary Fig. 1b). To further purify EVs obtained from B. cinerea liquid culture, the isolated EVs were subjected to sucrose gradient centrifugation as per the minimal information guidelines for studies of extracellular vesicles 2018 (MISEV2018) 48 . Analysis using NTA indicated that EVs were predominantly concentrated in sucrose fractions four and five with peak diameters of 93 nm and 94 nm, respectively (Fig. 1a). EVs were observed in both fractions and exhibited typical cupshaped structures as confirmed by negative staining and TEM analysis (Fig. 1b). The morphology and size distribution of the isolated B. cinerea EVs were comparable to EVs from other species, including plants and plant pathogenic fungi [31][32][33]49 , suggesting that B. cinerea has the ability to secrete EVs into the external environment.
Some tetraspanin proteins, such as CD63 in mammalian cells and tetraspanin 8 (TET8) in plant cells, are known to be markers for EVs 2,50 .
In B. cinerea, two tetraspanin coding genes, Punchless 1 (BcPLS1) and Tetraspanin 3 (BcTSP3), have been discovered 51 . Although these two proteins display limited amino acid sequence similarity, they share conserved structural hallmarks, including four transmembrane domains (TM1to TM4), a small extracellular loop (ECL1), an intracellular loop (ICL), and a large extracellular loop (ECL2) ( Supplementary  Fig. 2a, b). To determine whether these tetraspanin proteins are associated with B. cinerea EVs, both BcPLS1 and BcTSP3 proteins were tagged with a fluorescent protein YFP and inserted into the B. cinerea genome by homologous recombination. The EVs were isolated and separated using sucrose gradients from the liquid culture of both transgenic strains. As shown in Fig. 1c, BcPLS1 was found in the EV fractions three to five at the density of 1.11-1.19 g ml -1 and mainly concentrated at fraction four. However, BcTSP3 was hardly detected in isolated EV fractions, although it can be expressed in B. cinerea cells ( Fig. 1c and Supplementary Fig. 2c). Meanwhile, BcPLS1-YFP-positive EV signals can be observed under confocal microscopy (Fig. 1d).
Both transformed strains were then used to challenge wild-type Arabidopsis plants and samples were examined by confocal microscopy ten hours post-inoculation. As shown in Fig. 1e, BcPLS1-YFPlabeled vesicles were clearly observed outside of the fungal cells around infection sites. However, it was difficult to detect secreted vesicle signals from BcTSP3-YFP-infected plants. EVs were then isolated from BcPLS1-YFP or BcTSP3-YFP infected plants and separated using sucrose gradients. Both BcPLS1-YFP and Arabidopsis exosome marker TET8 were concentrated in the same fractions (Fig. 1f). To further confirm that B. cinerea secretes EVs during infection, BcPLS1-YFP or BcTSP3-YFP strains were used to inoculate Arabidopsis TET8-CFP transgenic plants. EVs were isolated from the apoplastic fluid of infected plants using sucrose gradients, and fraction four was examined under confocal microscopy. The EVs from BcPLS1-YFPinfected TET8-CFP plants exhibited two distinct fluorescent proteinlabeled signals (Fig. 1g). These results demonstrate that during infection, B. cinerea can secrete BcPLS1-positive EVs.

B. cinerea secretes small RNA effectors through EVs
Our previous work demonstrated that B. cinerea can transfer sRNA effectors into host plant cells to suppress host immunity and promote infection 3 . To study whether B. cinerea uses EVs to transport these sRNA effectors, we examined fungal sRNAs using sRNA stem-loop reverse transcription-polymerase chain reaction (RT-PCR) analysis in EVs isolated from B. cinerea liquid culture 52 . Three previously discovered sRNA effectors, Bc-siR3.1, Bc-siR3.2, and Bc-siR5, were detected in B. cinerea EVs isolated by sucrose gradient fractionation. These sRNAs were concentrated primarily in fractions four and five, which also contained high levels of BcPLS1 (Fig. 2a). Additionally, EVs from fraction four were used for a Micrococcal Nuclease (MNase) protection assay. As shown in Fig. 2b, the sRNA effectors can still be detected after MNase treatment unless vesicles were ruptured with Triton X-100 ( Fig. 2b and Supplementary Fig. 3). This result indicates that B. cinerea sRNAs are localized within and protected by EVs.
To assess the role of tetraspanin proteins in B. cinerea small RNA delivery, deletion mutants of BcPLS1 and BcTSP3 were generated by targeted gene replacement in wild-type B. cinerea strain B05 (Supplementary Fig. 4a). Two independent mutant strains of each gene Δbcpls1 and Δbctsp3 were complemented by expressing BcPLS1 and BcTSP3, respectively ( Supplementary Fig. 4a). On minimal medium (MM), but not potato dextrose agar, the growth rate of the Δbcpls1 strain was reduced compared to the wild-type and Δbctsp3 strains. (Supplementary Fig. 5). The spores of both mutants were collected and used to inoculate wild-type Arabidopsis plants to assess their virulence. The Δbcpls1 mutant strain showed a significant reduction in virulence with the lesion size being reduced by 84% compared with the wild-type strain (Fig. 2c). However, infection with Δbctsp3 resulted in lesions similar to the wild-type strain (Fig. 2c). To further understand the functions of BcPLS1 and BcTSP3 in EV-mediated sRNA secretion from B. cinerea, EVs were isolated from the culture supernatants of both the Δbcpls1 and Δbctsp3 strains and Bc-sRNA amounts were examined by real-time PCR and sRNA stem-loop RT-PCR. Compared with the wild-type strain, significantly lower levels of Bc-sRNAs were detected in Δbcpls1 EVs, while similar amounts of Bc-sRNAs were detected in Δbctsp3 EVs ( Fig. 2d and Supplementary Fig. 6a). Further, we found the EV amount released by Δbcpls1 was significantly reduced compared with wild-type and Δbctsp3 strains ( Supplementary Fig. 6b). This result indicates that BcPLS1 is important for EV-mediated sRNA secretion in B. cinerea.
To further confirm the function of BcPLS1 in B. cinerea virulence and EV secretion, we premixed Δbcpls1 spores with EVs isolated from c Western blot analysis of BcPLS1 and BcTSP3 in sucrose gradient fractions purified B. cinerea EVs from liquid culture. d B. cinerea EVs isolated from BcPLS1-YFP and BcTSP3-YFP liquid culture were examined by confocal microscopy. Scale bar, 10 μm. e BcPLS1-YFP labeled B. cinerea EVs were observed outside the fungal cells at the site of infection on Arabidopsis leaves. Samples were pretreated with 0.75 M sorbitol for 30 min to induce plasmolysis before imaging. Scale bars 10μm. f BcPLS1-YFP and BcTSP3-YFP were examined in sucrose gradient fractionated EVs that were isolated from BcPLS1-YFP or BcTSP3-YFP B. cinerea strains infected wildtype Arabidopsis using western blot. Arabidopsis TET8 was used as plant exosome control. g EVs isolated from BcPLS1-YFP or BcTSP3-YFP B. cinerea strains infected Arabidopsis TET8-CFP plants were purified using sucrose gradients, and the EVs from fraction four were examined using confocal microscopy. Scale bars, 10μm. Source data are provided as a Source Data file.  wild-type B. cinerea and used the mixture to infect Arabidopsis plants. As shown in Fig. 2e, EVs from wild-type B. cinerea can partially restore the virulence of Δbcpls1. The lesion size increased from 12% of the wildtype strain to 73% of the wild-type strain. This result demonstrates that the cargoes in B.cinerea EVs are important for fungal virulence.
To determine whether sRNAs in the EVs contribute to the fungal virulence, we premixed spores from a B. cinerea mutant dcl1dcl2 strain with EVs isolated from wild-type B. cinerea culture supernatants and then inoculated Arabidopsis plants. The dcl1dcl2 mutant cannot generate sRNA effectors, such as Bc-siR3.1, Bc-siR3.2, and Bc-siR5, and exhibits a very weak virulence phenotype 3 . However, when premixed with EVs from wild-type B. cinerea, which contain sRNA effectors, B. cinerea dcl1dcl2 mutant pathogenicity was dramatically recovered. The lesion size increased from 24% of the wild-type strain to 61% of the wild-type strain (Fig. 2f). We then further used EVs isolated from the dcl1dcl2 mutant to complement the phenotype of Δbcpls1. Results showed that B. cinerea dcl1dcl2 mutant EVs could only restore the virulence of Δbcpls1 from 18% of the wild-type strain to 32% of the wildtype strain ( Supplementary Fig. 7). This modest increase may be attributed to the other cargoes in the dcl1dcl2 EVs, because fungal EVs have been found to serve as a mode of transport for various virulencerelated proteins and toxic metabolites 53 . These results indicate that fungal DCL1/DCL2-generated sRNA effectors encapsulated in B. cinerea EVs play an important role in fungal virulence.

Clathrin-mediated endocytosis is involved in plant immunity against B. cinerea
In the mammalian system, multiple endocytic pathways are used to internalize EVs and their cargoes from various origins 54 . To determine whether endocytosis pathways are also involved in the uptake of B. cinerea EVs and their sRNA cargoes, we first treated wild-type Arabidopsis leaves with plant CME-specific inhibitor ES9-17 or CIE-specific inhibitor MβCD 55,56 . Vesicle internalization was strongly suppressed by ES9-17 but not by MβCD ( Supplementary Fig. 8a, c), suggesting that CME is likely the major pathway for EV uptake. However, we found that ES9-17 can also suppress the growth of B. cinerea ( Supplementary  Fig. 8b). So, the chemical treatment may cause a broad impact on both plants and fungi. Therefore, we chose genetic methods to ascertain the involvement of the CME pathway in the plant immune response to B. cinerea infection. We performed B. cinerea infection on two Arabidopsis CME pathway mutants, chc2 and ap2σ, which carry mutations in CLA-THRIN HEAVY CHAIN 2 and ADAPTOR 2 SIGMA SUBUNIT, respectively. Compared with wild-type plants, both chc2-1 and ap2σ mutants displayed reduced disease susceptibility to B. cinerea (Fig. 3a). The relative lesion size on chc2-1 and ap2σ mutants was significantly reduced by 60% and 73% compared with wild-type plants, respectively (Fig. 3a). Furthermore, we took advantage of inducible lines that expressed the C-terminal part of clathrin heavy chain (INTAM≫RFP-HUB1) or the clathrin coat disassembly factor Auxilin2 (AX2) (XVE≫AX2). Overexpression of both lines blocked CME in Arabidopsis 41,57 . After induction, both INTAM≫RFP-HUB1 and XVE≫AX2 lines showed significantly reduced susceptibility to B. cinerea infection (Fig. 3b, c). The relative lesion size on INTAM≫RFP-HUB1 and XVE≫AX2 was reduced by 45% and 59% compared with control plants. These results confirm that CME contributes to plant susceptibility to B. cinerea infection.
Next, we tested the possible function of the CIE pathway in plant immunity against B. cinerea infection. We examined the response of CIE-pathway mutants flot1/2, remorin 1.2, and remorin 1.3 after infection by wild-type B. cinerea. Because there is a close homolog of FLOT1 in Arabidopsis named FLOT2, we generated flot1/2 double mutants by knocking out FLOT2 in the flot1 mutant background using the Crispr-Cas9 system (Supplementary Fig. 9). Two independent lines were inoculated by B. cinerea, both of them showed a slightly more susceptible phenotype compared with wild-type (Fig. 3d). The homozygous knockout mutants of rem1.2 and rem1.3 T-DNA insertion lines were available and used for pathogen assays. No noticeable difference was observed between rem1.2 or rem1.3 and wild-type plants when challenged by B. cinerea spores (Fig. 3e). These results indicate that CME plays a more important role than CIE in plant susceptibility and the internalization of B. cinerea sRNAs into plant cells.
Bc-sRNA loading into host AGO1 and subsequent host target gene suppression is attenuated in CME mutants B. cinerea sRNAs can hijack the host plant RNAi machinery and silence plant immunity-related genes by binding to Arabidopsis Argonaute protein AGO1 3 . If CME is a critical factor in the internalization of EVs and Bc-sRNAs into plant cells, the deficiency of CME is likely to impact the level of sRNAs incorporated into the AGO1 protein. To determine whether CME is crucial for the uptake of B. cinerea sRNAs, we immunoprecipitated AGO1 from B. cinerea-infected wild-type Arabidopsis and CME mutants, including chc2-1 and ap2σ. As shown in Fig. 4a, the fungal sRNA effectors, Bc-siR3.1, Bc-siR3.2, and Bc-siR5, were all detected in the AGO1-associated fraction precipitated from infected wild-type plants. However, the amount of B. cinerea sRNAs loaded into AGO1 was significantly reduced by around 60% and 80% in chc2-1 and ap2σ, respectively, compared to wild-type plants. This observation was further supported by similar results obtained from INTAM≫RFP-HUB1 and XVE≫AX2 lines (Fig. 4b, c).
We also assessed whether CIE contributes to Bc-sRNA uptake by challenging flot1/2, rem1.2, and rem1.3 mutants with wild-type B.cinerea. Unlike CME mutants, the amount of AGO1-associated Bc-sRNAs in flot1/2, rem1.2, and rem1.3 mutants was similar to wild-type plants (Fig. 4d, e). As the total amount of AGO1 protein was not decreased in any of the CME and CIE mutants ( Supplementary Fig. 10), the decrease of Bc-sRNAs associated with AGO1 in CME mutants can be attributed to a reduction in the Bc-sRNA internalization of plant cells, rather than a decrease in the amount of AGO1 protein. Thus, CME is a key pathway for Bc-sRNA trafficking into host plant cells.
B. cinerea sRNA effectors have a suppressive effect on predicted host target genes during infection 3,58 . If CME is truly essential for crosskingdom trafficking of Bc-sRNAs, we expect to observe attenuated suppression of host target genes in CME mutants. Thus, we quantified the transcript levels of Bc-siR3.1 target gene At-PRXIIF, Bc-siR3.2 target genes At-MPK1 and At-MPK2, and Bc-siR5 target gene At-WAK, at 0 and 24 hours post-infection. As expected, the expression of these genes was suppressed in wild-type plants after 24 hours of infection. However, this suppressive effect was attenuated in the chc2-1, ap2σ, induced INTAM≫RFP-HUB1 and XVE≫AX2 mutants shown by no reduction in target gene transcript levels 24 hours after infection ( Fig. 4f and Supplementary Fig. 11a, b). We performed a similar experiment to explore the role of the CIE pathway in target gene suppression using the flot1/2, rem1.2, and rem1.3 mutants. Unlike CME mutants, all target genes were still significantly suppressed in the CIE mutants ( Supplementary Fig. 12a, b). These results again demonstrate that CME is the major pathway for Bc-sRNA uptake.

B. cinerea induces accumulation of clathrin-coated vesicles around infection sites
To further validate the role of CME in the internalization of B. cinerea sRNA effectors into host plant cells, Arabidopsis CLATHRIN LIGHT CHAIN 1 (CLC1)-GFP and CLATHRIN HEAVY CHAIN 2 (CHC2)-YFP transgenic plants were challenged by B. cinerea. Eight hours post inoculation, the infected plants were stained with the endocytic tracer FM4-64 and subjected to confocal microscopy to examine the role of endocytosis in plant response 38 . Numerous distinct fluorescently labeled endocytic vesicles were observed around B. cinerea infection sites, indicating the activation of endocytosis during B. cinerea infection (Fig. 5a, b). Meanwhile, we detected co-localization between CLC1-GFP or CHC2-YFP with the FM4-64 signal on vesicles at the infection sites, with co-localization rates of 52% and 60%, respectively (Fig. 5a, b).
These results suggest that B. cinerea infection induces the accumulation of CCVs around infection sites. In addition to clathrin, AP2 adaptor complexes are important components for CME in plant cells 59 . AP2A1-GFP plants were also treated with wild-type B. cinerea spores. Similar to CHC2-YFP and CLC1-GFP plants, clear colocalization (46%) was observed on the endocytic vesicles at the infection sites between AP2A1-GFP with FM4-64 (Fig. 5c). We also generated the YFP-tagged FLOT1 transgenic line and monitored the endocytosis through FM4-64 staining following inoculation with B. cinerea. As shown in Fig. 5d, endocytic FM4-64 signals can also be observed, but the percentage of colocalization (10%) between YFP-FLOT and FM4-64 was limited. These results further confirm that CME is more involved than CIE in B. cinerea infection of Arabidopsis.

B. cinerea EVs and sRNAs are associated with clathrin-coated vesicles
Unlike in animal cells, plant CCVs are not disassembled immediately after scission from the plasma membrane, which allows the isolation of CCVs by immunocapture 60 . We isolated plant CCVs from CLC1-GFP transgenic plants after infection with B. cinerea using a two-step procedure comprising differential centrifugation, followed by immunoaffinity purification with antibodies against GFP. GFP signal was clearly detected on the surface of GFP-TRAP agarose beads after incubation with isolated CCVs (Fig. 6a). To examine whether the immuno-captured CCVs were intact, the GFP-TRAP beads were subjected to scanning electron microscopy (SEM) analysis after pull down. Figure 6b shows that most of the immuno-captured CCVs were intact with typical cage structures on the surface of GFP-TRAP agarose beads. We further inoculated CLC1-GFP plants with the BcPLS1-mCherry B. cinerea strain. After ten hours of inoculation, colocalization of CLC1-GFP and BcPLS1-mCherry signals could be observed around the infection sites (Fig. 6c). Further, BcPLS1-mCherry can be detected in the CCV fractions isolated from infected plants (Fig. 6d). The Bc-sRNA effectors that we examined previously were also present in the immunopurified CCVs (Fig. 6e). These results provide conclusive evidence that B. cinerea EVs are responsible for transporting Bc-sRNAs and enabling their internalization through plant CME.

Discussion
Cross-kingdom RNAi is emerging as a critical regulatory mechanism across interaction systems. While recent studies show that plant and animal hosts utilize extracellular vesicles to protect and transport sRNAs to their fungal pathogens 2,5 , how fungi transported sRNAs to  CCVs from BcPLS1-mCherry infected CLC1-GFP plants using western blot. YFP plant was used as a negative control. e B. cinerea-secreted sRNAs can be detected in purified plant CCVs by RT-PCR. In d and e, the crude CCVs fraction obtained from B. cinerea-infected CLC-GFP or YFP plants through sucrose gradients was used for immunoprecipitation. Bc-siR17 and Bc-siR9 were negative controls. CLC1-GFP and YFP protein was detected by western blot using an anti-GFP antibody. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-023-40093-4 their hosts was still unknown. Here, we demonstrate that, just like their plant hosts, B. cinerea utilizes EVs to protect and transport sRNAs to plants, providing the first example of fungi utilizing EVs for crosskingdom RNA transport. Recently, the algal species Emiliania huxleyi was found to secrete vesicles containing sRNAs that influence viral infection and the population dynamics of interacting marine microorganisms 61,62 . Furthermore, bacterial EVs isolated from seawater were found to transport DNA transposons that facilitate horizontal gene transfer of nutrient-acquisition and bacteriophage-resistance genes among subpopulations of marine picocyanobacterium Prochlorococcus 63 . These studies demonstrate that EVs can survive in harsh environment and provide perfect protection for vulnerable RNA and DNA during transport, highlighting the underappreciated role of EVs in cross-kingdom/organismal communication, host-microbial interaction and microbial evolution 23 .
We show that plant cells internalize fungal EVs through clathrinmediated endocytosis. CME is one of the major pathways for plant uptake of environmental molecules, with extensive studies demonstrating its vital role in different biological processes 38,64 , including plant defense responses 39,41,[65][66][67] . While the plant CME pathway has recently been implicated in the uptake of protein effectors from fungal and oomycete pathogens 44,45 , we demonstrate here that it is also critical for the plant uptake of fungal sRNA effectors and fungal EVs. Although in mammals, intraorganismal EV internalization into recipient cells can occur through CME 68 , the mechanisms of internalization of foreign EVs from one organism to another in a different kingdom had never been reported until this study. We found that numerous CCVs accumulated around infection sites, indicating the activation of CME during B. cinerea infection. Additionally, the B. cinerea EV marker protein BcPLS1 is colocalized with plant CME component protein CLC1 during fungal infection and can be detected in the immunopurified plant CCVs. These findings strongly support that fungal EVs are internalized by the plant CME pathway. Furthermore, the EV-delivered sRNAs were also detected in the immunopurified CCVs, providing additional evidence for the role of plant CME in the uptake of fungal EV-delivered sRNAs.
These new insights into the mechanistic basis for cross-kingdom RNA communication between fungi and plants can help in the development of translational applications. For example, in B. cinerea and many other pathogens, such as Fusarium graminearum, Penicillium italicum, Colletotrichum gloeosporioides, and Valsa mali, sRNA effectors are generated by DCL proteins and used to promote virulence, and mutation in the DCL genes attenuates the virulence of these pathogens 3,69-72 . Consequently, targeting and silencing DCL and other components of the RNAi machinery, even transiently, has been proven to provide powerful protection against these pathogens by hampering their ability to synthesize sRNA effectors 16,[18][19][20][21][22] . Specifically, using spray-induced gene silencing (SIGS) to suppress the expression of both fungal DCL1 and DCL2 genes can effectively protect plants from infection by various pathogens, including fungal pathogens B. cinerea 16,[18][19][20] , F. graminearum 21 , and oomycete pathogen Plasmopara viticola 22 . Recent application of modern nanotechnology in SIGS 20,73-75 , especially the utilization of artificial nanovesicles that mimic EVs for RNA protection 19,73 , largely improves the efficacy of SIGS for crop protection. Here, by elucidating how sRNA effectors are transported from fungal pathogens into plants, we provide additional mechanistic targets for disease control. Specifically, we demonstrate that the EVassociated B. cinerea tetraspanin protein, BcPLS1, is an important protein for fungal EV secretion and virulence. Previous studies on PLS1 in both the B. cinerea T4 strain and Magnaporthe grisea showed that Δpls1 mutants exhibit weak virulence 76,77 , and the M. grisea PLS1 protein was found to be mainly expressed during infection and localized to appressorial membranes 77 . Consistently, we found the Δbcpls1mutant showed weak virulence on plants (Fig. 2c) and the BcPLS1 protein can be observed on the appressorial membranes (Fig. 1e). Furthermore, we detected BcPLS1 in the isolated B. cinerea EVs, and found that the delivery of B. cinerea sRNA effectors is compromised in the Δbcpls1 mutant, suggesting that BcPLS1-positive EVs are critical for sRNA effector transport. Thus, we have expanded the repetoire of effective genetic targets, such as PLS1, for RNAi-based anti-fungal strategies such as SIGS. Combining existing effective gene targets with PLS1 and other EV proteins targeting in SIGS will likely provide even more effective crop protection by targeting not only sRNA biogenesis, but also the trafficking mechanism of sRNA effectors from pathogens to plants.
Overall, our study helps to elucidate the underlying molecular mechanisms of EV-mediated sRNA trafficking during "cross-kingdom RNAi" and will further inform efforts in other organisms that also deliver sRNAs into interacting species. Given how widespread the phenomenon of cross-kingdom RNAi is in different branches of life, this will have strong rammifications for anti-pathogen measures in agriculture and human health.

Fungal strains and culture conditions
Botrytis cinerea strain B05.10 was used as a host strain for gene replacement and fluorescent labeling experiments, and as a wild-type (WT) control in all experiments. WT, mutants, and transgenic strains were all grown on complete media Potato Dextrose Broth with 1.5% Agar (PDA) at room temperature. The minimal medium (MM) utilized for measuring growth rate was prepared according to a previous study 78

EV isolation
EV isolation from fungal culture supernatant was performed as previously described 27 . In order to eliminate the possibility of contamination from the culture medium, the medium was initially ultracentrifuged at 100,000 g for 1 hour to eliminate any particles. Subsequently, the medium was filtered using a 0.22 μm filter prior to its use for B. cinerea culture. 10 6 B. cinerea spores were cultured for 48 hours at room temperature with shaking on the rocker in 100 ml of purified YEPD (0.3% yeast extract, 1% peptone, 2% glucose, pH 6.5) medium. The culture supernatant was collected by centrifugation at 3000 g for 15 mins at 4°C. The supernatant was then filtered through a 70 μm cell strainer to further remove cells. The cell-free supernatant was centrifuged at 10,000 g for 30 mins to remove smaller debris and a second round of centrifugation at 10,000 g for 30 mins was performed to ensure the removal of aggregates. The resulting supernatant was filtered through a 0.45 μm filter and ultracentrifuged at 100,000 g for 1 h to precipitate vesicles. Vesicles were washed once in phosphatebuffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4, 1.8 mM KH 2 PO 4 ), and the final pellets were resuspended in PBS.
EV isolation from infected plants was performed as previously described 2,49 . Apoplastic fluid was extracted from B. cinerea-infected Arabidopsis leaves by vacuum infiltration with infiltration buffer (20 mM MES, 2 mM CaCl 2 , 0.1 M NaCl, pH 6.0) and then centrifuged at 900 g for 10 mins. Cellular debris was further removed from the supernatant by centrifugation at 2000 g for 30 minutes, filtration through a 0.45 μm filter and finally centrifugation at 10,000 g for 30 mins. EVs were then collected by ultracentrifugation at 100,000 g for 1 hr and washed with infiltration buffer at 100,000 g for 1 hour.

Sucrose gradient separation of EVs
EVs from B. cinerea liquid culture and B. cinerea-infected Arabidopsis were purified by discontinuous sucrose density gradient centrifugation 25 . 10-90% sucrose stocks (w/v), including 10, 16, 22, 28, Article https://doi.org/10.1038/s41467-023-40093-4 34,40,46,52,58,64,70 and 90%, were prepared using infiltration buffer (20 mM MES, 2 mM CaCl2, 0.1 M NaCl, pH 6.0). The discontinuous gradient was prepared by layering 1 ml of each solution in a 15-ml ultracentrifuge tube. 100 μl EVs were premixed with 1 ml of 10% sucrose stock and loaded on top of the sucrose gradient. Then, samples were centrifuged in a swinging-bucket rotor for 16 h at 100,000 g, 4°C and six fractions (2 ml each) were collected from top to bottom. Collected fractions were transferred to new ultracentrifuge tubes and each sample was diluted to 12 ml using infiltration buffer, followed by a final centrifugation for 1 h at 100,000 g, 4°C to obtain pellets. The pellet was then resuspended in 50 μl infiltration buffer for further analysis.

Nanoparticle tracking analysis
A NanoSight NS300 fitted with a blue laser (405 nm) with NanoSight NTA software version 3.1 (Malvern Panalytical) was used to determine the size distribution and concentration of the B. cinerea EVs. EV samples were diluted 50 times using 0.22 μm filtered PBS and injected into the flow cell at a flow rate of 50 units. Four 60 second videos were taken for each sample and used to determine the size distribution and concentration of the EVs.

Transmission electron microscopy analysis
B. cinerea EVs were imaged using negative staining. Briefly, 10 μl of EVs were deposited onto 3.0 mm copper Formvar-carbon-coated grids (EMS) for 1 min and excess sample was removed from the grids using filter paper. The grids were then stained with 1% uranyl acetate for 30 s and blotted before a second round of staining with 1% uranyl acetate 2 min. The samples were air-dried before visualization with a Talos L120 transmission electron microscope, operated at 120 kV.

sRNA expression analyses
RNA was extracted using the Trizol extraction method for both total RNA and EV samples. sRNA RT-PCR was performed as previously described 2 . The PCR products were detected on a 12% PAGE gel. Quantitative PCR was performed with the CFX96 real-time PCR detection system (Bio-Rad) using SYBR Green mix (Bio-Rad). All primer sequences are provided in Supplementary Data 1.
To determine if sRNAs were localized within vesicles, EVs from sucrose gradient-purified fraction four were treated with 10 U of micrococcal nuclease (MNase) (Thermo Fisher) with or without Triton-X-100. For Triton-X-100 treatment, vesicles were incubated with 1% Triton-X-100 on ice for 30 minutes before nuclease treatments. Nuclease treatment was carried out at 37°C for 15 minutes followed by RNA isolation.

Transformation
The homologous recombination-based method was used to knock out B. cinerea genes as described previously 3 . The 5′-and 3′-regions of BcPLS1 and BcTSP3 were amplified and inserted into a modified pBluescript KS(+) plasmid (PBS-HPH) in which a hygromycin resistance cassette containing the hph gene of E. coli under the control of the trpC promoter of Aspergillus nidulans had been inserted between HindIII and SalI digestion sites. The full-length fragments containing the 5′and 3′-regions of each specific gene and the hph cassette were transformed into the protoplasts of wild-type B. cinerea. The transformants were purified by the isolation of a single spore on PDA containing 70 μg ml −1 hygromycin.
The complementary lines were generated by expressing the CDS of BcPLS1 or BcTSP3 in the Δbcpls1 or Δbctsp3 mutants, respectively. The CDS of both BcPLS1 and BcTSP3 were cloned separately into the pENTR vector (Life Technologies), then into the destination vector pBCC, which carries nourseothricin (NTC) N-acetyl transferase (NAT) conferring nourseothricin resistance. The specific primers 4-5-S and 4-3-A (Supplementary Data 1) were used to amplify dsDNA fragments containing promotor PtrpC, inserted genes, and NAT for B. cinerea transformation The positive transformants were selected on PDA plates with 50 μg ml -1 Nourseothricin Sulfate (Goldbio) and 70 μg ml −1 hygromycin.
The BcPLS1-YFP and BcTSP3-YFP strains were generated by amplifying and inserting the stop codon removed CDS region and 3' UTR region into the modified PBS-HPH, in which the YFP gene sequence has been inserted between BamHI and HindIII. The BcPLS1-mCherry was generated by inserting the stop codon removed CDS region and 3' UTR region into the modified PBS-HPH with the mCherry sequence inserted at the C terminal. The transformation and selection conditions were the same as the homologous recombination-based gene knock-out. All primers used are provided in Supplementary Data 1.

Plasmid construction
The vector for CHC2-YFP was constructed as follows: Arabidopsis genomic DNA was used as a template with primers CHC2-Pentr-F and CHC2-Pentr-R to amplify the whole sequence of CHC2. The final PCR product was cloned into pENTR/SD/D-TOPO (Life Technologies), and then into the destination vector pEarleyGate 101 by LR reactions (Life Technologies). For the YFP-FLOT1 construct, the full-length CDS of FLOT1 was cloned into the pENTR/SD/D-TOPO vector (Life Technologies), then into the destination vector pEARLYGATE 104 for YFP tagging (Life Technologies). For the TET8-CFP construct, the full-length CDS of TET8 was cloned into the pENTR/SD/D-TOPO vector (Life Technologies), then into the destination vector pEARLYGATE 102 for CFP tagging (Life Technologies). For the construct to express YFP, the YFP sequence was amplified from pEARLYGATE 101 and then cloned into pENTR/SD/D-TOPO vector and finally into pEARLYGATE 100 (Life Technologies). Primer sequences are listed in Supplementary Data 1.

Chemical solutions and treatments
ES9-17 (Sigma Aldrich) was dissolved in DMSO to create a 30 mM stock solution. mβCD (Sigma Aldrich) was dissolved in deionized water to create a 200 mM stock solution. 4-Hydroxytamoxifen (Sigma Aldrich) was dissolved in DMSO to make a 2 mM stock solution. Estradiol (Sigma Aldrich) was dissolved in DMSO to make a 5 mM stock solution. For the chemical inhibitor treatments, Arabidopsis plant leaves were incubated in liquid 1/2 Murashige and Skoog (MS) medium with 30 μM or 10 mM mβCD for 24 hours. DMSO and deionized water were used as controls. To induce the expression of RFP-HUB1 or AX2, 2 μM 4-Hydroxytamoxifen or 5 μM estradiol was used to pretreat the plant leaves in liquid 1/2 MS medium for 24 hours.

CCV purification
The clathrin-coated vesicle isolation procedure was adapted from previously established protocols 83,84 . 50 grams of Arabidopsis leaves were homogenized for 1 min in 2 volumes of resuspension buffer (100 mM MES, pH 6.4, 3 mM EDTA, 0.5 mM MgCl2, 1 mM EGTA, protease inhibitors (Roche)) using a Waring Blender. The homogenate was squeezed through eight layers of cheesecloth and three layers of Miracloth (Sigma). Then the homogenate was centrifuged in four increments to obtain a microsomal pellet: 500 g, 5 min; 5000 g, 10 min; 20,000 g, 30 min; 100,000 g, 60 min. The pellet was then resuspended in 6 ml resuspension buffer. Then the resuspended pellet was loaded on top of the discontinuous sucrose gradients, which were made from three sucrose stocks (10%, 35%, and 50%) in the resuspension buffer. The gradient was composed of 1 ml of 50% sucrose, 5 ml of 35% sucrose, and 3 ml of 10% sucrose in a Beckman 14 × 95 mm Ultra-Clear centrifuge tube. The gradients were centrifuged at 116,000 g 50 min. Samples were collected from 10% and 10/35% interface and resuspended in the resuspension buffer to make the sucrose concentration lower than 5%. The samples were then centrifuged at 200,000 g for 50 min, and the pellets were collected as crude CCVs.
Further immunoisolation of CCVs was performed as follows: GFP-Trap® Agarose (ChromoTek) was equilibrated in the resuspension buffer. Plant crude CCVs from CLC1-GFP and YFP were incubated with the GFP-Trap® Agarose for 2 hr at 4°C. After, beads were washed three times with resuspension buffer. The RNA from purified CCVs was extracted using TRIzol Reagent (Invitrogen) following immunoisolation. Subsequently, the immunoisolated CCVs on GFP-Trap Agarose beads were further analyzed by a scanning electron microscope (SEM).

Confocal microscopy
Arabidopsis seedlings were imaged on a Leica TCS SP5 confocal microscope (Leica Microsystems). Briefly, leaves were infiltrated with 10 μM FM4-64 dye for 30 mins before examination. Samples were imaged using a 40x water immersion dip-in lens mounted on a Leica TCS SP5 confocal microscope (Leica Microsystems). The excitation wavelength was 488 nm for GFP, 514 nm for YFP, and 559 nm for FM4-64. Emission was detected at 500-550 nm for GFP and YFP, 570-670 nm for FM4-64. The colocalization percentage was processed by means of the ImageJ (Fiji) software package. More specifically, the regions of interest (ROI) were selected to include vesicles accumulated around B. cinerea infection area. The total area of FM4-64 (red) and overlapped area (yellow) were measured and used for colocalization calculation. The average of three independent values was presented in the figures. The method for intracellular/PM signal intensity ratio quantification was described previously 55 . Non-saturated images were converted in ImageJ (Fiji) to 8-bit and regions of interest (ROIs) were selected based on the PM or cytosol localization. Intensity values per ROI of PM and intracellular signals were measured and used for calculation.

Pathogen assay
B. cinerea was cultured on PDA plates for 10 days at room temperature before spores were collected and diluted into inoculation medium (0.1% sabouraud maltose broth buffer, Difco) for a final concentration of 10 5 spores per mL. Four-week-old plants were then inoculated by applying a single 10 μl droplet of B. cinerea per leaf. Photos were taken 2 days after inoculation and analyzed by ImageJ software to measure the lesion size.
For pathogen assay using a mixture of WT or Bc-dcl1/dcl2 B. cinerea EVs with Δbcpls1 and Bc-dcl1/dcl2, 10 6 B. cinerea spores were cultured for 48 hours at room temperature with shaking on the rocker in 100 ml of YEPD (0.3% yeast extract, 1% peptone, 2% glucose, pH 6.5) medium. B. cinerea EVs were isolated and purified using sucrose gradients as described above, then EVs from fraction four were resuspended in 100 μl PBS. The spores were collected and diluted into 2 x inoculation medium (0.2% sabouraud maltose broth buffer, Difco) for a final concentration of 2 × 10 5 spores per mL. Equal volumes of EVs and spores from mutant strains were mixed. Four-week-old plants were then inoculated by applying a single 10 μl droplet of EVs and spores mixture per leaf. Equal volumes of EVs and 2 x inoculation medium were mixed to inoculate plants as the negative control. Photos were taken 2 days after inoculation and analyzed by ImageJ software to measure the lesion size.

Real-time RT-PCR
Total RNA was extracted from plant tissues using TRIzol and the concentration was measured at 260 nm using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Equal amounts of total RNA were treated with DNase I enzyme (Roche, Basel, Switzerland) and converted into first-strand cDNA using SuperScript III (Invitrogen, Carlsbad, CA). The cDNA was then used to check target gene expression by real-time RT-PCR with gene-specific primers. Primer sequences are listed in Supplementary Data 1.

AGO1 immunoprecipitation
Arabidopsis AGO1 IP was performed with 5 g fresh leaves collected 24 hr after spray inoculation with B. cinerea. Tissues were collected and ground in liquid nitrogen before resuspension in 5 ml of extraction buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% NP40, proteinase inhibitor cocktail; Sigma). The extract was then precleared by incubation with 50 ul of Protein A-agarose beads (Roche) at 4°C for 30 min. The precleared extract was incubated with 10 ul of AGO1-specific antibody for 6 hours. 50 ul of Protein A-agarose beads (Roche) was then added to the extract before overnight incubation. Immunoprecipitates were washed three times (5 min each) in wash buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% Triton x-100, proteinase inhibitor cocktail; Sigma). RNA was extracted from the bead-bound AGO1 with TRIzol reagent (Invitrogen). Proteins were detected by boiling the bead-bound AGO1 in SDS-loading buffer before running in a SDS-PAGE gel.

Scanning electron microscope (SEM)
The structure of CCVs isolated using GFP-Trap Agarose was examined using field emission scanning electron microscopy Nova NanoSEM 450 (FEI) at 15 kV. The samples were washed three times with PBS (pH 7.2) and fixed with 2.5% glutaraldehyde overnight at 4°C. Samples were dehydrated with gradient alcohol solutions (10%, 30%, 50%, 70%, 80%, and 90%) for 10 min, 100% ethanol for 20 min, and 100% acetone for 30 min. Subsequently, samples were dried by CO 2 under critical point. Samples were sputter coated with gold and fixed on the stab using carbon tape before observation and imaging.

Statistical analysis
The statistical analyses were performed using analysis of variance (ANOVA), Dunnett's multiple comparisons test, and unpaired twotailed Student's t-test using GraphPad Prism (9.0.2). The statistical tests and n numbers, including sample sizes or biological replications, are described in the figure legends.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The data that support the findings of this study are available within the paper, Supplementary Information, and Source Data. Source data are provided with this paper.